Microbial contaminants including nontuberculous mycobacteria (NTM) in metalworking fluids (MWFS) have been implicated in occupational respiratory illnesses. Our broader goals are to develop and apply practical DNA-based approaches for real-time detection, quantitation, and identification of mycobacteria in MWF for understanding the prevalent strains and their survivability in commercial MWFS. The specific aims are (i) to develop polymerase chain reaction (PCR)-based protocols for real-time detection and quantitation of NTM in water-based MWF; (ii). to PCR screen field samples of different commercial formulations of water-based MWFs for NTM followed by strain-specific identification of the NTM isolates using molecular typing methods; (iii) to investigate viability of the identified NTM strains in MWF using quantitative PCR. A Mycobacterium genus-specific PCR-based method will be optimized for real-time detection of NTM in water-based MWF. The method will be used as a basis for developing a quantitative PCR protocol for quantitation of NTM in field MWF samples. NTM strains will be isolated from MWF samples in use across the industries and in selected plants linked with hypersensitivity pneumonitis cases. The isolates will be identified at the species level by 16S ribosomal gene amplicon sequencing and at the strain level based on genome fingerprinting using pulsed field gel electrophoresis (PFGE) or rapid amplification of polymorphic DNA-PCR (RAPD-PCR) technique. Effect of strain type and biocide load on viability of the selected strains in MWF will be investigated in simulated as well as field conditions using quantitative PCR. The resulting information will help NORA's objectives by providing practical methods for microbial exposure assessment and database for developing intervention strategies related to MWF exposures.